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Semester 3: Molecular Biology And Microbial Genetics
DNA Structure - Salient features of double helix, forms of DNA. Denaturation and renaturation. DNA topology Supercoiling, linking number, topoisomerases
DNA Structure
The DNA molecule is made up of two long strands that form a double helix. Each strand is composed of nucleotides, which consist of a sugar, a phosphate group, and a nitrogenous base. The two strands are held together by hydrogen bonds between the complementary bases (adenine with thymine, and guanine with cytosine).
Salient Features of Double Helix
The double helix structure allows for the compact storage of genetic information. It has a major groove and a minor groove which proteins bind to for cellular processes. The strands are anti-parallel, meaning they run in opposite directions, which is crucial for replication and transcription.
Forms of DNA
DNA can exist in several forms, including A, B, and Z DNA. The B form is the most common in physiological conditions, while A DNA is more compact and Z DNA has a left-handed twist and is formed under certain conditions.
Denaturation and Renaturation
Denaturation is the process where DNA strands separate due to the breaking of hydrogen bonds, often caused by heat or chemical treatments. Renaturation is the reverse process where separated strands can reanneal under suitable conditions.
DNA Topology
DNA topology refers to the physical state of DNA. It can discuss the way DNA coils and folds within the cell. The structure can be altered by enzymatic processes during replication and transcription.
Supercoiling
Supercoiling refers to the additional twist in the DNA double helix beyond the normal helical structure. This affects the accessibility of the DNA for replication and transcription.
Linking Number
The linking number is a topological property of DNA that describes the number of times one strand wraps around the other. It is a crucial factor in understanding DNA structure and functionality.
Topoisomerases
Topoisomerases are enzymes that manage DNA supercoiling and tangling. They introduce or remove supercoils by cutting the DNA strands, allowing them to unwind or reseal, which is critical during DNA replication and transcription.
DNA organization in prokaryotes, viruses, eukaryotes. Replication of DNA in prokaryotes and eukaryotes - Bidirectional and unidirectional replication, semi-conservative and semi-discontinuous replication
DNA organization and replication in prokaryotes, viruses, and eukaryotes
DNA organization in prokaryotes
Prokaryotes generally have a circular DNA molecule that is not enclosed within a nucleus. Their DNA exists in a region called the nucleoid. Prokaryotic DNA is compacted by supercoiling and associated with proteins called histone-like proteins.
DNA organization in viruses
Viruses can have either DNA or RNA as their genetic material. Viral DNA can be circular or linear, single-stranded or double-stranded. Virus DNA is encapsulated within a protein coat known as a capsid, and some viruses have an additional lipid envelope.
DNA organization in eukaryotes
Eukaryotic DNA is linear and organized into chromosomes. It is located within the nucleus and associated with histones to form nucleosomes, which further compact to form chromatin. The organization is critical for gene regulation and expression.
Replication of DNA in prokaryotes
Prokaryotic DNA replication is typically bidirectional, beginning at a single origin of replication. The process includes initiation, elongation, and termination, utilizing enzymes such as DNA polymerase.
Replication of DNA in eukaryotes
Eukaryotic DNA replication also occurs bidirectionally but involves multiple origins of replication on each chromosome. It is more complex than prokaryotic replication and includes additional enzymes and regulatory mechanisms.
Bidirectional and unidirectional replication
Bidirectional replication proceeds in two directions away from the origin, seen in both prokaryotes and eukaryotes. Unidirectional replication proceeds only in one direction, which is less common in cellular organisms.
Semi-conservative replication
In semi-conservative replication, each new DNA molecule contains one original strand and one newly synthesized strand. This mechanism ensures genetic continuity across generations of cells.
Semi-discontinuous replication
Semi-discontinuous replication refers to the way replication occurs on the leading and lagging strands. The leading strand is synthesized continuously, while the lagging strand is synthesized in short segments called Okazaki fragments, requiring multiple initiation events.
Mechanism of DNA replication enzymes involved DNA polymerases, DNA ligase, primase
Mechanism of DNA replication enzymes involved: DNA polymerases, DNA ligase, primase
Overview of DNA Replication
DNA replication is a biological process that occurs in all living organisms to copy their DNA. It is essential for cell division, allowing for genetic continuity and variation.
DNA Polymerases
DNA polymerases are key enzymes responsible for synthesizing new DNA strands by adding nucleotides complementary to the template strand. There are several types of DNA polymerases, each with specific functions during replication. For example, DNA polymerase III is the primary enzyme for DNA synthesis in prokaryotes.
DNA Ligase
DNA ligase is an enzyme that plays a crucial role in joining Okazaki fragments on the lagging strand during DNA replication. It catalyzes the formation of phosphodiester bonds between adjacent nucleotides, sealing any nicks in the DNA backbone.
Primase
Primase is an RNA polymerase that synthesizes a short RNA primer, which serves as a starting point for DNA synthesis. This primer is essential for DNA polymerases to initiate the replication process.
Replication Fork
The replication fork is the area where the DNA double helix is unwound to allow for replication. Multiple enzymes work at the replication fork, including helicase, which unwinds the DNA, and single-strand binding proteins, which stabilize the unwound strands.
Lagging and Leading Strands
During replication, the leading strand is synthesized continuously, whereas the lagging strand is synthesized in small segments known as Okazaki fragments. This requires the coordinated action of primase, DNA polymerase, and DNA ligase.
Regulation and Fidelity
The activity of DNA replication enzymes is carefully regulated to ensure high fidelity in DNA synthesis, minimizing the errors that could lead to mutations.
DNA replication modes - rolling circle, D-loop modes
DNA replication modes
Rolling Circle Replication
Rolling circle replication is a method of DNA replication that is used primarily by certain viruses and plasmids. In this mode, a circular DNA molecule has one strand cleaved, allowing the other strand to be continuously replicated in a rolling manner. The process begins with a single nick in the circular DNA, leading to the displacement of the 5' end while the 3' end serves as a template for replication. This results in the formation of a long concatamer of repeated DNA sequences which can be cleaved into individual genomic units.
D-loop Replication
D-loop replication is a type of replication primarily observed in mitochondrial and certain plasmid genomes. In D-loop replication, a segment of the double-stranded DNA is unwound, creating a displacement loop (D-loop). This allows for the synthesis of one strand while the other strand remains intact. The displaced strand can then serve as a template for the synthesis of a complementary strand, resulting in the formation of multiple D-loops. This mechanism is crucial for the replication of mitochondrial DNA where rapid synthesis is necessary.
Transcription in Prokaryotes. Concept of transcription. RNA Polymerases - prokaryotic and eukaryotic
Transcription in Prokaryotes
Concept of Transcription
Transcription is the process by which genetic information in DNA is copied into RNA. In prokaryotes, transcription occurs in the cytoplasm and is directly coupled with translation.
RNA Polymerases in Prokaryotes
Prokaryotic cells primarily use a single type of RNA polymerase that is responsible for synthesizing all types of RNA: mRNA, tRNA, and rRNA. This enzyme binds to the promoter region of the DNA to initiate transcription.
RNA Polymerases in Eukaryotes
Eukaryotic cells contain three different RNA polymerases. RNA Polymerase I synthesizes rRNA (except 5S rRNA), RNA Polymerase II synthesizes mRNA and some snRNA, while RNA Polymerase III synthesizes tRNA, 5S rRNA, and other small RNAs.
Comparison of Prokaryotic and Eukaryotic Transcription
Unlike prokaryotic transcription which occurs in the cytoplasm and lacks a nucleus, eukaryotic transcription occurs in the nucleus. Eukaryotic transcription also requires multiple transcription factors and is more complex due to the presence of introns and exons.
Importance of Prokaryotic Transcription
Prokaryotic transcription is critical for gene expression and regulation in bacteria. It allows for rapid responses to environmental changes and is essential for the synthesis of proteins required for cell survival.
General transcription factors in eukaryotes
General transcription factors in eukaryotes
Overview of Eukaryotic Transcription
Eukaryotic transcription occurs in the nucleus and involves multiple steps: initiation, elongation, and termination. It requires a complex set of proteins, including general transcription factors, to ensure accurate transcription of DNA into RNA.
General Transcription Factors
General transcription factors are essential proteins that facilitate the binding of RNA polymerase to the promoter region of a gene. Key factors include TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, each playing a unique role in the transcription initiation process.
Mechanism of Action
General transcription factors work by recognizing specific sequences in the core promoter region, forming a pre-initiation complex. This complex recruits RNA polymerase II, allowing the transcription process to commence.
Regulatory Elements
In addition to general transcription factors, eukaryotic transcription is regulated by various elements such as enhancers and silencers. These elements can significantly influence the binding of general transcription factors and the overall transcriptional output.
Post-Transcriptional Modifications
After transcription initiation, eukaryotic mRNA undergoes several modifications, including capping, polyadenylation, and splicing. While these processes are not directly related to general transcription factors, they are crucial for the maturation of RNA and its subsequent translation into proteins.
Implications of Dysregulation
Dysregulation of general transcription factors can lead to various diseases, including cancer. Understanding these factors is critical for the development of targeted therapies and gene-editing technologies.
Distinction between transcription processes in prokaryotes versus eukaryotes
Overview of Transcription
Transcription is the first step of gene expression, where a segment of DNA is copied into RNA by RNA polymerase. Prokaryotes and eukaryotes exhibit distinct transcription processes.
Prokaryotic Transcription
In prokaryotes, transcription occurs in the cytoplasm. The process is simpler, with a single type of RNA polymerase. Transcription and translation can occur simultaneously, and prokaryotic mRNA is often polycistronic, meaning it can code for multiple proteins.
Eukaryotic Transcription
In eukaryotes, transcription occurs in the nucleus and involves three different RNA polymerases. Eukaryotic mRNA undergoes extensive processing, including capping, polyadenylation, and splicing, before it is transported to the cytoplasm for translation.
Regulation of Transcription
Prokaryotic transcription is regulated primarily at the initiation step via promoters and transcription factors. Eukaryotic transcription is more complex, involving a range of regulatory elements, enhancers, and a variety of transcription factors that interact with RNA polymerase and mediator complexes.
Transcription Terminators
In prokaryotes, termination of transcription can occur via intrinsic mechanisms or through rho-dependent mechanisms. In eukaryotes, transcription termination is usually linked to the processing of the pre-mRNA and is more intricate, often involving specific sequences in the DNA.
Translation in prokaryotes and eukaryotes - Translational machinery - ribosome structure in prokaryotes and eukaryotes, tRNA structure and processing
Translation in prokaryotes and eukaryotes
Translational Machinery
Translation involves decoding mRNA into a protein. In both prokaryotes and eukaryotes, ribosomes play a central role. Prokaryotic ribosomes are 70S, composed of 50S and 30S subunits, while eukaryotic ribosomes are 80S, composed of 60S and 40S subunits.
Ribosome Structure in Prokaryotes
Prokaryotic ribosomes consist of a 30S small subunit and a 50S large subunit. The 30S subunit is responsible for binding mRNA, while the 50S subunit facilitates peptide bond formation during translation. The ribosomal RNA (rRNA) in these subunits is essential for their structure and function.
Ribosome Structure in Eukaryotes
Eukaryotic ribosomes are larger and more complex than prokaryotic ribosomes. The 40S subunit binds to mRNA, and the 60S subunit contains the peptidyl transferase center. Eukaryotic ribosomes have a greater number of proteins and rRNA components, aiding in their functional diversity.
tRNA Structure
Transfer RNAs (tRNAs) are essential for translating mRNA into proteins. Each tRNA molecule carries a specific amino acid at one end and has an anticodon region that base pairs with the corresponding codon on mRNA. This structure allows for accurate translation.
tRNA Processing
tRNA undergoes several processing steps, including splicing and addition of a CCA tail at the 3' end, which is critical for amino acid attachment. In prokaryotes, tRNA is often processed during transcription, while in eukaryotes, processing occurs post-transcriptionally.
Inhibitors of protein synthesis in prokaryotes and eukaryotes
Inhibitors of protein synthesis in prokaryotes and eukaryotes
Overview of Protein Synthesis
Protein synthesis is the process through which cells generate proteins. It involves two main stages: transcription and translation. In prokaryotes, transcription and translation occur simultaneously in the cytoplasm, while in eukaryotes, transcription occurs in the nucleus and translation in the cytoplasm.
Types of Protein Synthesis Inhibitors
Protein synthesis inhibitors can be classified into two categories based on their target organisms: inhibitors of prokaryotic protein synthesis and inhibitors of eukaryotic protein synthesis.
Inhibitors of Prokaryotic Protein Synthesis
Key inhibitors include: 1. Aminoglycosides (e.g., streptomycin): Bind to the 30S ribosomal subunit, causing misreading of mRNA. 2. Tetracyclines: Block the attachment of aminoacyl-tRNA to the ribosome's A site. 3. Macrolides (e.g., erythromycin): Bind to the 50S subunit, inhibiting peptide chain elongation. 4. Chloramphenicol: Inhibits peptide bond formation by binding to the 50S subunit.
Inhibitors of Eukaryotic Protein Synthesis
Key inhibitors include: 1. Cycloheximide: Inhibits translocation during translation in eukaryotic ribosomes. 2. Puromycin: Resembles aminoacyl-tRNA and causes premature termination of translation. 3. Ricin: A highly toxic protein that inactivates the 60S ribosomal subunit, blocking protein synthesis. 4. Anisomycin: Inhibits peptide bond formation in eukaryotic ribosomes.
Mechanisms of Action
Different inhibitors have distinct mechanisms of action, which target various stages of protein synthesis, such as initiation, elongation, and termination, affecting the overall process differently in prokaryotes and eukaryotes.
Clinical Applications
Many protein synthesis inhibitors are utilized as antibiotics to treat bacterial infections. Understanding the differences in how these inhibitors affect prokaryotes versus eukaryotes is essential for the development of targeted therapies.
Resistance Mechanisms
Bacteria can develop resistance to protein synthesis inhibitors through various mechanisms, including enzymatic modification of the drug, alteration of ribosomal targets, and efflux pumps. This necessitates ongoing research into new inhibitors and alternative treatments.
Overview of regulation of gene
Overview of regulation of gene
Gene Regulation Basics
Gene regulation refers to the processes that control the expression of genes. This can occur at various stages including transcription, RNA processing, translation, and post-translational modifications. Understanding these mechanisms is crucial for insights into cellular differentiation, development, and responses to environmental changes.
Types of Gene Regulation
There are several types of gene regulation, including transcriptional, translational, and post-translational regulation. Transcriptional regulation involves proteins that bind to DNA and influence RNA polymerase activity. Translational regulation affects the efficiency of mRNA translation into protein, while post-translational regulation involves modifications made to proteins after they have been synthesized.
Regulatory Elements
Regulatory elements such as promoters, enhancers, silencers, and insulators play critical roles in gene regulation. Promoters are regions where RNA polymerase binds to initiate transcription. Enhancers increase the likelihood of transcription, while silencers repress it. Insulators act as barriers to prevent the interaction of enhancers with promoters.
Epigenetic Regulation
Epigenetic mechanisms, including DNA methylation and histone modifications, influence gene expression without altering the underlying DNA sequence. These changes can be stable and heritable, impacting gene regulation in development, environmental responses, and disease.
Regulation in Prokaryotes vs Eukaryotes
Gene regulation differs significantly between prokaryotic and eukaryotic organisms. In prokaryotes, regulation often occurs at the transcriptional level through operons, while in eukaryotes, regulation is multi-layered with more complex interactions among regulatory proteins, non-coding RNAs, and chromatin dynamics.
Applications of Gene Regulation
Understanding gene regulation has important implications in biotechnology, medicine, and agriculture. Techniques such as CRISPR-Cas9 have emerged from the study of gene regulation, allowing for precise editing of genes to study functions or correct mutations.
